Membrane docking geometry and target lipid stoichiometry of membrane-bound PKCα C2 domain: a combined molecular dynamics and experimental study

Abstract

Protein kinase Cα (PKCα) possesses a conserved C2 domain (PKCα C2 domain) that acts as a Ca(2+)-regulated membrane targeting element. Upon activation by Ca(2+), the PKCα C2 domain directs the kinase protein to the plasma membrane, thereby stimulating an array of cellular pathways. At sufficiently high Ca(2+) concentrations, binding of the C2 domain to the target lipid phosphatidylserine (PS) is sufficient to drive membrane association; however, at typical physiological Ca(2+) concentrations, binding to both PS and phosphoinositidyl-4,5-bisphosphate (PIP(2)) is required for specific plasma membrane targeting. Recent EPR studies have revealed the membrane docking geometries of the PKCα C2 domain docked to (i) PS alone and (ii) both PS and PIP(2) simultaneously. These two EPR docking geometries exhibit significantly different tilt angles relative to the plane of the membrane, presumably induced by the large size of the PIP(2) headgroup. The present study utilizes the two EPR docking geometries as starting points for molecular dynamics simulations that investigate atomic features of the protein-membrane interaction. The simulations yield approximately the same PIP(2)-triggered change in tilt angle observed by EPR. Moreover, the simulations predict a PIP(2):C2 stoichiometry approaching 2:1 at a high PIP(2) mole density. Direct binding measurements titrating the C2 domain with PIP(2) in lipid bilayers yield a 1:1 stoichiometry at moderate mole densities and a saturating 2:1 stoichiometry at high PIP(2) mole densities. Thus, the experiment confirms the target lipid stoichiometry predicted by EPR-guided molecular dynamics simulations. Potential biological implications of the observed docking geometries and PIP(2) stoichiometries are discussed.

Fig. 1. MD simulations of the PKCα C2 domain on a lipid bilayer

Snapshots from MD simulations of the complex composed of a C2 domain and (a) a bilayer lacking PIP₂; (b) a bilayer containing one PIP₂ molecule; and (c) a bilayer containing two PIP₂ molecules. These simulations were termed (−)PIP₂, (+)PIP_2(one), and (+)PIP_2(two) respectively. Lipids are shown in gray, PS headgroups are shown as small pink spheres, and the lysine cluster formed by the side chains of K197, K199, K209, and K211 are blue spheres. Nearby K205 is also highlighted by blue spheres. In (+)PIP_2(one) system, thePIP₂ headgroup is shown as red spheres; in (+)PIP_2(two) system, the two PIP2 headgroups are colored differently as red and orange spheres. In each case the simulation was initiated by placing the C2 domain (PDB entry 1DSY, drawn as a ribbon) with two bound calcium ions (yellow spheres) on the surface of a 1:1 POPC/POPS mixed lipid bilayer. The C2 domain was positioned in the center of the bilayer patch with an initial penetration depth and angle relative to the bilayer surface that matched the docking geometry previously determined by EPR analysis. As indicated, zero, one or two PIP₂ molecules were placed near the lysine cluster. This initial system was equilibrated as described in Methods, then a 30 ns unconstrained MD simulation was carried out during which a representative single frame snapshot was taken for the figure. The vector (orange arrow) operationally defines the long axis of the core β-sandwich (from Ca²⁺501 to the Cα atom of N206). The plane (dashed line) indicates the average location of the lipid backbone phosphate groups. In the absence of PIP₂ the long axis of the membrane-bound C2 domain is oriented nearly parallel to the bilayer surface with a tilt angle of 7° ± 2°. The binding of one PIP₂ to the lysine cluster tilts the domain away from the membrane plane, yielding a tilt angle of 37° ± 2°, while the binding of two PIP₂ yields an intermediate tilt angle of 30° ± 1°. Each tilt angle and its standard deviation was calculated by averaging the mean tilt angle of six time blocks obtained by breaking the 30 ns simulation into 5 ns sections.

Fig. 2. Temporal stability of the docking geometry

Shown are the time-varying tilt angles of the three different C2 domain-membrane complexes during each 30 ns simulation. Details as in Figure 1 legend. (−)PIP₂, black line; (+)PIP_2(one), green line; and (+)PIP_2(two), blue line.

Fig. 3. Lipids contacting the bound C2 domain

Snapshots from the (−)PIP₂ and (+)PIP_2(two) simulations, highlighting the 18 and 14 lipid headgroups contacting the C2 domain, respectively. The lipid footprint of the (+)PIP_2(one) simulation (not shown) is indistinguishable from that of the (+)PIP_2(two) simulation. The two PIP₂ headgroups are colored differently as red and orange spheres, other headgroups in contact with the C2 domain are grey spheres.

Fig. 4. Binding of PKCα C2 domain to PIP₂-containing membranes

A standard protein-to-membrane FRET assay was employed to quantitate membrane-bound C2 domain at 25° C; . (a) Determination of PIP₂ stoichiometry. Target membranes containing a fixed PIP₂ mole fraction were titrated into a fixed concentration of C2 domain. Conditions were chosen to drive high-affinity PIP₂ binding, such that the titration yielded a linear increase in membrane-associated C2 domain until all protein was bound and a plateau was achieved. The intersection of best-fit straight lines for the linear increase and plateau regions represents the saturation point, yielding 2.1 PIP₂ molecules per C2 domain in this representative example. The target membranes employed were sonicated unilamellar vesicles containing 10 mole % PIP₂ in a lipid mixture mimicking plasma membrane inner leaflet. Samples contained 1.0 μM C2 domain and 10 μM free Ca²⁺ in a physiological buffer: 25 mM HEPES at pH 7.4 with KOH, 140 mM KCl, 15 mM NaCl, 0.5 mM MgCl₂, and sufficient EDTA to generate the desired free [Ca²⁺] (see Methods). (b) Analysis of PIP₂ cooperativity. Target membranes containing the indicated mole fractions of PIP₂ were added to yield a fixed total lipid concentration in a fixed concentration of C2 domain. Conditions were chosen to simulate a cytoplasmic Ca²⁺ signal. Nonlinear least squares best-fit of the resulting membrane binding curve with the Hill equation yields 5.1 ± 0.4 for this representative example, but the interpretation of this apparent positive cooperativity is complex (see Discussion). Samples contained 60 μM total lipid (see (a) for composition), 0.5 μM C2 domain and 1 μM free Ca²⁺ in physiological buffer (see (a)).